Vesicle Release Sites Research Articles

Developmental refinement of the active zone nanotopography and axon wiring at the somatosensory thalamus

Functional refinement of neural circuits is a crucial developmental process in the brain. However, how synaptic maturation and axon wiring proceed cooperatively to establish reliable signal transmission is unclear. Here, we combined nanotopography of release machinery at the active zone (AZ), nanobiophysics of neurotransmitter release, and single-neuron reconstruction of axon arbors of lemniscal fibers (LFs) in the developing mouse somatosensory thalamus. With development, the cluster of Cav2.1 enlarges and translocates closer to vesicle release sites inside the bouton, and LFs drastically shrink their arbors and form larger boutons on the perisomatic region of target neurons. Experimentally constrained simulations show that the nanotopography of mature synapses enables not only rapid vesicular release but also reliable transmission following repetitive firing. Sensory deprivation impairs the developmental shift of molecular nanotopography and axon wiring. Thus, we uncovered the cooperative nanotopographical and morphological mechanisms underlying the developmental establishment of reliable synaptic transmission.

Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

Lead exposure induced transgenerational developmental neurotoxicity by altering genome methylation in Drosophila melanogaster

Heavy metal toxicity is a significant global health concern, with particular attention given to lead (Pb) exposure due to its adverse effects on cognitive development, especially in children exposed to low concentrations. While Pb neurotoxicity has been extensively studied, the analysis and molecular mechanisms underlying the transgenerational effects of Pb exposure-induced neurotoxicity remain poorly understood. In this study, we utilized Drosophila, a powerful developmental animal model, to investigate this phenomenon. Our findings demonstrated that Pb exposure during the developmental stage had a profound effect on the neurodevelopment of F0 fruit flies. Specifically, we observed a loss of correlation between the terminal motor area and muscle fiber area, along with an increased frequency of the β-lobe midline crossing phenotype in mushroom bodies. Western blot analysis indicated altered expression levels of synaptic vesicle proteins, with a decrease in Synapsin (SYN) and an increase in Bruchpilot (BRP) expression, suggesting changes in synaptic vesicle release sites. These findings were corroborated by electrophysiological data, showing an increase in the amplitude of evoked excitatory junctional potential (EJP) and an increase in the frequency of spontaneous excitatory junctional potential (mEJP) following Pb exposure. Importantly, our results further confirmed that the developmental neurotoxicity resulting from grandparental Pb exposure exhibited a transgenerational effect. The F3 offspring displayed neurodevelopmental defects, synaptic function abnormalities, and repetitive behavior despite lacking direct Pb exposure. Our MeDIP-seq analysis further revealed significant alterations in DNA methylation levels in several neurodevelopmental associated genes (eagle, happyhour, neuroglian, bazooka, and spinophilin) in the F3 offspring exposed to Pb. These findings suggest that DNA methylation modifications may underlie the inheritance of acquired phenotypic traits resulting from environmental Pb exposure.

An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity.

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

Computational modeling of trans-synaptic nanocolumns, a modulator of synaptic transmission.

Understanding synaptic transmission is of crucial importance in neuroscience. The spatial organization of receptors, vesicle release properties and neurotransmitter molecule diffusion can strongly influence features of synaptic currents. Newly discovered structures coined trans-synaptic nanocolumns were shown to align presynaptic vesicles release sites and postsynaptic receptors. However, how these structures, spanning a few tens of nanometers, shape synaptic signaling remains little understood. Given the difficulty to probe submicroscopic structures experimentally, computer modeling is a useful approach to investigate the possible functional impacts and role of nanocolumns. In our in silico model, as has been experimentally observed, a nanocolumn is characterized by a tight distribution of postsynaptic receptors aligned with the presynaptic vesicle release site and by the presence of trans-synaptic molecules which can modulate neurotransmitter molecule diffusion. In this work, we found that nanocolumns can play an important role in reinforcing synaptic current mostly when the presynaptic vesicle contains a small number of neurotransmitter molecules. Our work proposes a new methodology to investigate in silico how the existence of trans-synaptic nanocolumns, the nanometric organization of the synapse and the lateral diffusion of receptors shape the features of the synaptic current such as its amplitude and kinetics.

Mechanisms and Consequences of Cerebellar Purkinje Cell Disinhibition in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), the most common form of childhood muscular dystrophy, is caused by mutations in the dystrophin gene. In addition to debilitating muscle degeneration, patients display a range of cognitive deficits thought to result from the loss of dystrophin normally expressed in the brain. While the function of dystrophin in muscle tissue is well characterized, its role in the brain is still poorly understood. The highest expression of dystrophin in the mouse brain is in cerebellar Purkinje cells (PCs), where it colocalizes with GABAA receptor clusters. Using ex vivo electrophysiological recordings from connected molecular layer interneuron (MLI)-PC pairs, we investigated changes in inhibitory synaptic transmission caused by dystrophin deficiency. In male mdx mice (which lack long-form dystrophin), we found that responses at MLI-PC pairs were reduced by ∼60% because of both decreased quantal response amplitude and a reduced number of functional vesicle release sites. Using electron microscopy, we found significantly fewer and smaller anatomically defined inhibitory synapses contacting the soma of PCs in mdx mice, suggesting that dystrophin may play a critical role in synapse formation and/or maintenance. Functionally, we found reduced MLI-evoked pauses in PC firing in acute slices. In vivo recordings from awake mdx mice showed increased sensory-evoked simple spike firing in positively modulating PCs, consistent with reduced feedforward inhibition, but no change in negatively modulating PCs. These data suggest that dystrophin deficiency in PCs disrupts inhibitory signaling in the cerebellar circuit and PC firing patterns, potentially contributing to cognitive and motor deficits observed in mdx mice and DMD patients.SIGNIFICANCE STATEMENT Duchenne muscular dystrophy (DMD) is primarily characterized by progressive muscle weakening caused by genetic mutations in the gene for dystrophin. Dystrophin is also normally expressed in the CNS, and DMD patients experience a range of nonprogressive cognitive deficits. The pathophysiology of CNS neurons resulting from loss of dystrophin and the function of dystrophin in neurons are still poorly understood. Using cerebellar PCs as a model, we found that the loss of dystrophin specifically disrupts the number and strength of inhibitory synaptic connections, suggesting that dystrophin participates in formation and/or maintenance of these synapses. This work provides insight into the function of dystrophin in the CNS and establishes neuronal and synaptic dysfunction, which may underlie cognitive deficits in DMD.

Interaction of Munc13-1 and RIM that contributes to the formation/maintenance of synaptic vesicle release sites

Interaction of Munc13-1 and RIM that contributes to the formation/maintenance of synaptic vesicle release sites

Interactions of active zone proteins with Munc13-1 that contribute to the formation of synaptic vesicle release sites

Interactions of active zone proteins with Munc13-1 that contribute to the formation of synaptic vesicle release sites

Molecular and functional architecture of striatal dopamine release sites

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.

Mesophasic organization of GABAA receptors in hippocampal inhibitory synapses.

Information processing in the brain depends on specialized organization of neurotransmitter receptors and scaffolding proteins within the postsynaptic density. However, how these molecules are organized in situ remains largely unknown. In this study, template-free classification of oversampled sub-tomograms was used to analyze cryo-electron tomograms of hippocampal synapses. We identified type-A GABA receptors (GABAARs) in inhibitory synapses and determined their in situ structure at 19-Å resolution. These receptors are organized hierarchically: from GABAAR super-complexes with a preferred inter-receptor distance of 11 nm but variable relative angles, through semi-ordered, two-dimensional receptor networks with reduced Voronoi entropy, to mesophasic assembly with a sharp phase boundary. These assemblies likely form via interactions among postsynaptic scaffolding proteins and receptors and align with putative presynaptic vesicle release sites. Such mesophasic self-organization might allow synapses to achieve a 'Goldilocks' state, striking a balance between stability and flexibility and enabling plasticity in information processing.

α2δ-2 Protein Controls Structure and Function at the Cerebellar Climbing Fiber Synapse.

α2δ proteins (Cacna2d1-4) are auxiliary subunits of voltage-dependent calcium channels that also drive synapse formation and maturation. Because cerebellar Purkinje cells (PCs) predominantly, if not exclusively, express one isoform of this family, α2δ-2 (Cacna2d2), we used PCs as a model system to examine roles of α2δ in excitatory synaptic function in male and female Cacna2d2 knock-out (KO) mice. Whole-cell recordings of PCs from acute cerebellar slices revealed altered climbing fiber (CF)-evoked complex spike generation, as well as increased amplitude and faster decay of CF-evoked EPSCs. CF terminals in the KO were localized more proximally on PC dendrites, as indicated by VGLUT2+ immunoreactive puncta, and computational modeling demonstrated that the increased EPSC amplitude can be partly attributed to the more proximal location of CF terminals. In addition, CFs in KO mice exhibited increased multivesicular transmission, corresponding to greater sustained responses during repetitive stimulation, despite a reduction in the measured probability of release. Electron microscopy demonstrated that mutant CF terminals had twice as many vesicle release sites, providing a morphologic explanation for the enhanced glutamate release. Though KO CFs evoked larger amplitude EPSCs, the charge transfer was the same as wild-type as a result of increased glutamate reuptake, producing faster decay kinetics. Together, the larger, faster EPSCs in the KO explain the altered complex spike responses, which degrade information transfer from PCs and likely contribute to ataxia in Cacna2d2 KO mice. Our results also illustrate the multidimensional synaptic roles of α2δ proteins.SIGNIFICANCE STATEMENT α2δ proteins (Cacna2d1-4) regulate synaptic transmission and synaptogenesis, but coexpression of multiple α2δ isoforms has obscured a clear understanding of how various α2δ proteins control synaptic function. We focused on roles of the α2δ-2 protein (Cacna2d2), the deletion of which causes cerebellar ataxia and epilepsy in mice and humans. Because cerebellar Purkinje cells (PCs) only express this single isoform, we studied excitatory climbing fiber synaptic function onto PCs in Cacna2d2 KO mice. Using optical and electrophysiological analysis, we provide a detailed description of the changes in PCs lacking α2δ-2, and provide a comprehensive mechanistic explanation for how functional synaptic phenotypes contribute to the altered cerebellar output.

Rapid regulation of vesicle priming explains synaptic facilitation despite heterogeneous vesicle:Ca2+ channel distances.

Chemical synaptic transmission relies on the Ca2+-induced fusion of transmitter-laden vesicles whose coupling distance to Ca2+ channels determines synaptic release probability and short-term plasticity, the facilitation or depression of repetitive responses. Here, using electron- and super-resolution microscopy at the Drosophila neuromuscular junction we quantitatively map vesicle:Ca2+ channel coupling distances. These are very heterogeneous, resulting in a broad spectrum of vesicular release probabilities within synapses. Stochastic simulations of transmitter release from vesicles placed according to this distribution revealed strong constraints on short-term plasticity; particularly facilitation was difficult to achieve. We show that postulated facilitation mechanisms operating via activity-dependent changes of vesicular release probability (e.g. by a facilitation fusion sensor) generate too little facilitation and too much variance. In contrast, Ca2+-dependent mechanisms rapidly increasing the number of releasable vesicles reliably reproduce short-term plasticity and variance of synaptic responses. We propose activity-dependent inhibition of vesicle un-priming or release site activation as novel facilitation mechanisms.

Spatiotemporal dynamics of multi-vesicular release is determined by heterogeneity of release sites within central synapses

A synaptic active zone (AZ) can release multiple vesicles in response to an action potential. This multi-vesicular release (MVR) occurs at most synapses, but its spatiotemporal properties are unknown. Nanoscale-resolution detection of individual release events in hippocampal synapses revealed unprecedented heterogeneity among vesicle release sites within a single AZ, with a gradient of release probability decreasing from AZ center to periphery. Parallel to this organization, MVR events preferentially overlap with uni-vesicular release (UVR) events at sites closer to an AZ center. Pairs of fusion events comprising MVR are also not perfectly synchronized, and the earlier event tends to occur closer to AZ center. The spatial features of release sites and MVR events are similarly tightened by buffering intracellular calcium. These observations revealed a marked heterogeneity of release site properties within individual AZs, which determines the spatiotemporal features of MVR events and is controlled, in part, by non-uniform calcium elevation across the AZ.

In Vitro Configuration of Munc13-1 Bridging of Phospholipid Bilayers at Resting Conditions

Neurotransmitter release is an intricate process involving multiple proteins that govern synaptic vesicle exocytosis. While SNAREs drive membrane fusion, chaperone proteins regulate their function, ensuring fusion happens in a precise and controllable manner. Munc13-1 is an elongated, multi-domain protein participating in docking through plasma membrane interactions via the N-terminal C1, C2B domains and vesicle tethering via the C-terminal C2C domain and SNARE priming by opening Syntaxin-1A, thus promoting SNARE-pin formation. In addition, it was recently proposed that Munc13-1 is a core component of molecular architecture at the synaptic vesicle release site due to its ability to form a supramolecular assembly (clusters). Stability of these clusters could imply ordered spatial arrangement of Munc13-1 molecules but to date, no structural evidence has been reported. Here we report the spatial arrangement of the truncated version of Munc13-1 consisting of C1, C2B, MUN (Δ1408-1442, EF) and C2C domains (Munc13L) on the surface of negatively charged phospholipid membranes. Using cryo-tomography, we found that Munc13L forms hexagonal 2D crystal in Ca2+ free conditions, bridging two membrane bilayers. Subtomograms extracted from the observed crystal were aligned and averaged to yield a 3D map at 14 Å resolution. Fitting of existing crystal structures of C1, C2B and MUN domains revealed that Munc13L exists in two conformational states in the crystal. One form is extended, connecting two bilayers to form a triskelion-like structure by the interaction of C-terminal ends of MUN domains, while the other form is supporting the extended form using the interaction of its C2C domain with MUN region of the supported molecule. These interactions are novel and require further investigation to determine the physiological role and its transformation when activated by calcium.

Variance analysis as a tool to predict the mechanism underlying synaptic plasticity

BackgroundThe strength of synaptic transmission onto a neuron depends on the number of functional vesicle release sites (N), the probability of vesicle release (Pr), and the quantal size (Q). Statistical tools based on the quantal model of synaptic transmission can be used to acquire information on which of these parameters is the source of plasticity. However, quantal analysis depends on assumptions that may not be met at central synapses. New methodWe examined the merit of quantal analysis to extract the mechanisms underlying synaptic plasticity by applying binomial statistics on the variance in amplitude of postsynaptic currents evoked at Schaffer collateral-CA1 (Sc-CA1) synapses in mouse hippocampal slices. We extend this analysis by combining the conventional inverse square of the coefficient of variation (1/CV2) with the variance-to-mean ratio (VMR). ResultsThis method can be used to assess the relative, but not absolute, contribution of N, Pr and Q to synaptic plasticity. The changes in 1/CV2 and VMR values correctly reflect experimental modifications of N, Pr and Q at Sc-CA1 synapses. Comparison with existing methodsWhile the 1/CV2 depends on N and Pr, but is independent of Q, the VMR is dependent on Pr and Q, but not on N. Combining both allows for a rapid assessment of the mechanism underlying synaptic plasticity without the need for additional electrophysiological experiments. ConclusionCombining the 1/CV2 with the VMR allows for a reliable prediction of the relative contribution of changes in N, Pr and Q to synaptic plasticity.

Nanoscale molecular landscape of the synaptic vesicle release site in the hippocampus

Nanoscale molecular landscape of the synaptic vesicle release site in the hippocampus

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (τm) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Glucose and lactate as metabolic constraints on presynaptic transmission at an excitatory synapse.

Synapses have high energy demands which increase during intense activity. We show that presynaptic terminals can utilise extracellular glucose or lactate to generate energy to maintain synaptic transmission. Reducing energy substrates induces a metabolic stress: presynaptic ATP depletion impaired synaptic transmission through a reduction in the number of functional synaptic vesicle release sites and a slowing of vesicle pool replenishment, without a consistent change in release probability. Metabolic function is compromised in many pathological conditions (e.g. stroke, traumatic brain injury and neurodegeneration). Knowledge of how synaptic transmission is constrained by metabolic stress, especially during intense brain activity, will provide insights to improve cognition following pathological insults. The synapse has high energy demands, which increase during intense activity. Presynaptic ATP production depends on substrate availability and usage will increase during activity, which in turn could influence transmitter release and information transmission. We investigated transmitter release at the mouse calyx of Held synapse using glucose or lactate (10, 1 or 0mm) as the extracellular substrates while inducing metabolic stress. High-frequency stimulation (HFS) and recovery paradigms evoked trains of EPSCs monitored under voltage-clamp. Whilst postsynaptic intracellular ATP was stabilised by diffusion from the patch pipette, depletion of glucose increased EPSC depression during HFS and impaired subsequent recovery. Computational modelling of these data demonstrated a reduction in the number of functional release sites and slowed vesicle pool replenishment during metabolic stress, with little change in release probability. Directly depleting presynaptic terminal ATP impaired transmitter release in an analogous manner to glucose depletion. In the absence of glucose, presynaptic terminal metabolism could utilise lactate from the aCSF and this was blocked by inhibition of monocarboxylate transporters (MCTs). MCT inhibitors significantly suppressed transmission in low glucose, implying that lactate is a presynaptic substrate. Additionally, block of glycogenolysis accelerated synaptic transmission failure in the absence of extracellular glucose, consistent with supplemental supply of lactate by local astrocytes. We conclude that both glucose and lactate support presynaptic metabolism and that limited availability, exacerbated by high-intensity firing, constrains presynaptic ATP, impeding transmission through a reduction in functional presynaptic release sites as vesicle recycling slows when ATP levels are low.

Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. Most HD studies have focused on degeneration of the CNS. We previously discovered that skeletal muscle from transgenic R6/2 HD mice is hyperexcitable due to decreased chloride and potassium conductances. The progressive and early onset of these defects suggest a primary myopathy in HD. In this study, we examined the relationship between neuromuscular transmission and skeletal muscle hyperexcitability. We used an ex vivo preparation of the levator auris longus muscle from male and female late-stage R6/2 mice and age-matched wild-type controls. Immunostaining of the synapses and molecular analyses revealed no evidence of denervation. Physiologically, we recorded spontaneous miniature endplate currents (mEPCs) and nerve-evoked EPCs (eEPCs) under voltage-clamp, which, unlike current-clamp records, were independent of the changes in muscle membrane properties. We found a reduction in the number of vesicles released per action potential (quantal content) in R6/2 muscle, which analysis of eEPC variance and morphology indicate is caused by a reduction in the number of vesicle release sites (n) rather than a change in the probability of release (prel). Furthermore, analysis of high-frequency stimulation trains suggests an impairment in vesicle mobilization. The depressed neuromuscular transmission in R6/2 muscle may help compensate for the muscle hyperexcitability and contribute to motor impersistence.SIGNIFICANCE STATEMENT Recent evidence indicates that Huntington's disease (HD) is a multisystem disorder. Our examination of neuromuscular transmission in this study reveals defects in the motor nerve terminal that may compensate for the muscle hyperexcitability in HD. The technique we used eliminates the effects of the altered muscle membrane properties on synaptic currents and thus provides hitherto the most detailed analysis of synaptic transmission in HD. Clinically, the striking depression of neurotransmission we found may help explain the motor impersistence in HD patients. Therapies that target the highly accessible peripheral nerve and muscle system provide a promising new avenue to lessen the debilitating motor symptoms of HD.